We are investigating hepatocarcinogen binding to drug metabolizing enzymes in vivo and in vitro. 2-Acetylaminofluorene (2-AAF) feeding produces the PN antigen effect in male rat liver microsomes, possibly through covalent binding to epoxide hydrase and/or other proteins of liver endoplasmic reticulum such as the cytochrome P450's (NADPH dependent monooxygenases). Covalent binding of radiolabeled 2-AAF to microsomal proteins has been demonstrated and is being studied using enzyme purification and analytical SDS gel electrophoresis-radioautography. The site(s) of addition will be estimated by peptide mapping, amino acid sequencing and specific chemical and enzymatic reactions. We are quantitating the PN antigen effect. Optimum in vitro incubations have allowed immuno-, enzymatic, and SDS-electrophoretic characterization of the release of epoxide hydrase from hyperplastic nodule and hepatoma microsomes. Epoxide hydrase has been found in the cytosolic fractions of nodule and hepatoma cells, but not in normal liver cells. A new polypeptide has also been detected, and is another "PN Antigen". The chemical characterization of the alteration in nodule epoxide hydrase versus normal enzyme is being studied. Methods for a high-yield purification of normal epoxide hydrase have been found and have shown to be reliable. Nodule and hepatoma microsomal epoxide hydrase will be purified by this procedure; and carbohydrate content of each species will be quantitated. Also, the presence of bound 2-AAF (even in hepatoma epoxide hydrase) will be assayed for by; a) a comparison of two-dimensional electrophoresis of polypeptide cleavage products, b) immunological techniques (by antiserum to 2-AAF labeled hydrase, back-adsorbed with normal hydrase), and c) radiometric analysis.